I. Field of the Invention
The present invention relates to the general field of biochemical assays and separations, generally classified in U.S. Patent Class 435.
II. Description of the Prior Art
Interest in nucleic acid purification has increased with human trials of plasmid-based vaccines (e.g., for influenza, HIV, and malaria) and therapeutics (e.g., insulin and vascularization promoters) as well as the steady expansion of DNA sequencing activities. (references 1 and 2) This invention embodies a rapid, scaleable, nuclease-free (preferably RNAse free), cost effective method of nucleic acid purification using selective precipitation by compaction agents.
Prior Art will include the following:
1. Parasrampuria, D. and Hunt, A., (1998), Therapeutic issues in gene therapy; part 1: vectors. Biopharm. 11:38-45.
2. Anderson, F., (1998), Human Gene Therapy. Nature. 392: 25-30.
3. Horn, N. A., Meek, J. A., Budahazi, G., and Marquet M. 1995. Cancer gene therapy using plasmid DNA: purification of DNA for human clinical trials. Human Gene Therapy. 6:565-573.
4. Gosule, L. C. and Schellman, J. A., (1976), Compact form of DNA induced by spermidine. Nature. 259:333-335.
5. Arscott, P. G., Li, A. Z., and Bloomfield, V. A., (1990), Condensation of DNA by trivalent cations. 1. Effects of DNA length and topology on the size and shape of condensed particles. Biopolymers. 30:619-630.
6. Wilson, R. W. and Bloomfield, V. A., (1979), Counter-ion induced condensation of deoxyribonucleic acid. A light scattering study. Biochemistry. 18:2192-2196.
7. Bednar, J., Furrer, P., Stasiak, A., Dubochet, J., Egelman, E. H., and Bates, A. D., (1994), The twist, writhe and overall shape of supercoiled DNA change during counterion-induced transition from a loosely to a tightly interwound superhelix: possible implications for DNA structure in vivo. Journal of Molecular Biology. 235:825-847.
8. Rolland, A., (1998), From genes to gene medicines: recent advances in nonviral gene delivery. Critical Review of Therapeutic Drug Carrier Systems. 15:143-198.
9. Hoopes, B. C. and McClure, W. R., (1981), Studies on the selectivity of DNA precipitation by spermine. Nucleic Acids Research. 9:5493-5504.
10. Sambrook, J., Fritsch, E. F., and Maniatis, T., (1989), Molecular cloning, a laboratory manual. Second edition, Cold Spring Harbor Laboratory Press.
11. Horn, N., Marquet, M., Meek, J., and Budahazi, G., (1996), Process for reducing RNA concentration in a mixture of biological material using diatomaceous earth. U.S. Pat. No. 5,576,196.
12. Lev, Z., (1987), A procedure for large-scale isolation of RNA-free plasmid and phage DNA without the use of RNAse. Analytical Biochemistry. 160:332-336.
13. Drevin, I., Larsson, L., and Johansson, B. L., (1989), Column performance of Q-Sepharose HP in analytical- and preparative-scale chromatography. Journal of Chromatography. 477:337-344.
14. Horn et al; U.S. Pat. No. 5,707,812, Purification of Plasmid DNA During Column Chromatography, which is understood to teach addition of short chain polymeric alcohol to promote isolation of plasmid DNA.
15. Hubert, P., and Dellacherie, E., (1980), Use of water-soluble biospecific polymers for the purification of proteins, Journal of Chromatography, 184, 325-333.
16. Irwin, J. A., and Tipton, K. F., (1995), Affinity precipitation: a novel approach to protein purification, Essays in Biochemistry, 29, 137-156.
17. Widom, J., and Baldwin, R. L., (1983), Monomolecular condensation of xcex-DNA induced by Cobalt Hexammine, Biopolymers, 22, 1595-1620.
18. Nunn, C. S., and Neidle, S. 1996. The high resolution crystal structure of the DNA decamer d(AGGCATGCCT). J. Mol. Biol. 256:340-351.
19. Kieft, J. S. and Tinoco, I. 1997. Solution structure of a metal-binding site in the major groove of RNA complexed with cobalt (III) hexammine. Structure. 5(5):713-721.
20. Pitulle, C., Hedenstierna, K. O., and Fox, G. E. 1995. A novel approach for monitoring genetically engineered microorganisms by using artificial, stable RNAs. Applied Environmental Microbiology. 61(10): 3661-3666.
21. Setterquist, R. A, Smith, G. K., Oakley, T. H., Lee, Y. H., and Fox, G. E. 1996. Sequence, overproduction and purification of Vibrio proteolyticus ribosomal protein L18 for in vitro and in vivo studies. Gene. 183(1-2):237-242.
22. Yang, Y. and Fox, G. E. 1996. An Archaea 5S rRNA analog is stablely expressed in Escherichia coli. Gene. 168: 81-85.
23. Sioud, M. and Drlica, K. 1991. Prevention of human immunodeficiency virus type 1 integrase expression in Escherichia coli by a ribozyme. Proc. Natl. Acad. Sci. USA 88:7303-7307.
24. Couture, L. A. and Stinchcomb, D. T. 1996. Anti-gene therapy: the use of ribozymes to inhibit gene function. TIG. 12(12):510-514.
25. Christoffersen, R. E., and Marr J. J., (1995), Ribozymes as human therapeutic agents, Journal of Medicinal Chemistry, 38(12), 2023-2037.
26. Weiss, B., Davidkova, G. and Zhou L. W., (1999), Antisense RNA gene therapy for studying and modulating biological processes, Cell. Mol. Life Sci., 55, 334-358.
27. Kumar, M. and Carmichael, G. G., (1998), Antisense RNA: function and fate of duplex RNA in cells of higher eukaryotes, Microbiology and Molecular Biology Reviews, 62(4), 1415-1434.
28. Matthews, H. R., (1993), Polyamines, chromatin structure and transcription, BioEssays, 15(8), 561-566.
29. Hedemstierna, K. O. F., Lee, H. Y., Yang, Y., and Fox, G. E, (1993), A prototype stable RNA identification cassette for monitoring plasmids of genetically engineered microorganisms. System. Appl. Microbiol. 16, 280-286.
30. Pitulle, C., Dsouza, L., and Fox, G. E. 1997. A low molecular weight artificial RNA of unique size with multiple probe target regions. System. Appl. Microbiol. 20:133-136.
31. Uchiyama, S., Imamura, T., Nagai, S., and Konishi, K. 1981. Separation of low molecular weight RNA species by high-speed gel filtration. J. Biochem. 90:643-648.
32. Lee, K. M. and Marshall, A. G. 1986. High-speed preparative-scale separation and purification of ribosomal 5S and 5.8S RNA""s via Sephacryl S-300 gel filtration chromatography. Preparative Biochemistry 16(3):247-258.
33. Hori, S. and Ohtani, S. 1990. Separation of high-molecular mass RNAs by high-performance liquid chromatography on hydroxyapatite. Journal of Chromatography. 515:611-619.
34. Fair, W. R., and Wehner, N., (1971) Antibacterial action of spermine: effect on urinary tract pathogens, Applied Environmental Microbiology, 21(1), 6-8
35. Scopes, R. K., (1993) Protein purification: principles and practice, Springer-Verlag, 379 pages.
36. Blackburn, G. M., and Gait, M. J., (1996), Nucleic Acids in Chemistry and Biology, Oxford University Press, pages 337-346.
37. Saenger, W., (1988), Principles of Nucleic Acid Structure, Springer-Verlag, pages 432-434.
38. Ma, C., Sun, L., and Bloomfield, V. A., (1995), xe2x80x9cCondensation of Plasmids Enhanced by Z-DNA Conformation of d(CG)n Insertsxe2x80x9d, Biochemistry, vol. 34(11), 3521-3528.
39 U.S. Pat. No. 5,622,822, to Tobias et al, Issued Apr. 22, 1997, (Assigned Johnson and Johnson), Methods for capture and selective release of nucleic acids using polyethyleneimine and an anionic phosphate ester surfactant and amplification of same teaches that nucleic acids can be made available for amplification or other treatment after lysis by contacting the lysate with polyethyleneimine to form a precipitate with the nucleic acids. The nucleic acids are then released from the precipitate by contact with a strong base, and the released nucleic acids are kept in solution with an anionic phosphate ester surfactant.
II. Problems Presented by Prior Art
Most current methods of plasmid separation are relatively time-consuming and require the use of adsorbents, toxic substances, nucleases, and/or filtration media to separate plasmid from protein, genomic DNA, endotoxins and especially the abundant RNA present in cell lysates.
This technique offers several important improvements over current methods: no RNAse and/or other enzymes are used, the technique requires no chromatographic medium, and the technique is directly scaleable if larger quantities of plasmid DNA are needed.
Also with the use of different compaction agents, different types of nucleic acids can be separated from the same mixture. The invention can separate different types of RNA and DNA as long as some secondary structure is present.
In addition RNA can be fractionated based on molecular weight via selective precipitation.
Different compaction agents also have different affinities for different nucleic acids. For example hexammine cobalt has a higher affinity for RNA than the polyamine spermidine so multiple step selective precipitations have been developed to help separate nucleic acids as quickly as possible.
The method can also be used for parallel purification of a large number of samples (mini-preps) and is readily adaptable to automation (robotics).
In another embodiment, the invention also provides a method for making a biochemical assay by hybridizing a labeled probe to a target (e.g. chromosomal DNA, oligonucleotides, Ribosomal RNA, tRNA, plasmid, aptamer, viral RNA), and thereafter precipitating the probe/target complex with compaction agents. For example, preparing a mixture containing chromosomal DNA, plasmid, ribosomal RNA, and labeled oligonucleotides, then heating the mixture of nucleic acids above their melting temperature (if the hybridization site is buried within secondary structure) and thereafter precipitating the probe and the target).
In another embodiment, the invention also provides a method for separating a nucleic acid-binding protein from a mite containing the protein and its nucleic acid binding partner and other components, by precipitating the bound nucleic acid, carrying the associated protein into the precipitate, from which it may optionally be further purified. For example, a selected protein might be isolated from cultured human cells containing both the protein and a DNA sequence to which the protein binds, by making a lysate from the cells and precipitating the DNA, producing a precipitate enriched in both the DNA target sequence and in the binding protein.
Bioseparations, especially separation of RNA from DNA or vice versa, are conventionally accomplished in bench scale or larger pilot plants in which a fermentation is carried out to produce cell mass which is lysed, then exposed to filtration and the use of nucleases to reduce unwanted nucleic acid populations (e.g. the use of ribonuclease (RNAse) in plasmid purification). Generally, after these initial solution phase purification steps, the effluent products are further purified by chromatographic columns (e.g. anion-exchange or size-exclusion chromatography), often with samples being analyzed and results subjected to quality control feedback techniques. Such procedures can take a day or more for a single run or batch on a single mixture, assuming the optimum conditions, concentrations, etc. The present invention permits the separation of dozens of feed mixtures in a single set-up, often in less time than required for a single separation by conventional methods. Further, when practiced in its preferred embodiments, the invention can sharply reduce the production costs (costs per milligram of purified DNA product produced).
In addition, the labeled probe precipitation embodiment offers a new method for hybridization assays without the use of radiolabeled probes or the use of solid supports. Using compaction precipitation, when a tagged probe (e.g. fluoresceinated, radioactively tagged, etc.) is added to a solution containing its target a double stranded nucleic acid is formed and this new structured hybrid can be selectively precipitated while the single stranded probe will be left in solution.
In addition, the nucleic acid/binding protein coprecipitation embodiment offers a new method of identifying and/or separating nucleic acid-binding proteins from cells expressing them. Using compaction precipitation, these proteins can be selectively precipitated away from other proteins, producing a significant degree of selective enrichment without the need to prepare costly affinity adsorbent matrices.
Summarizing, preferred embodiments include the assay, the protein purification, and selectivity for DNA precipitation over RNA, and isolation of RNA by first precipitating DNA, then separately precipitating RNA in a second step.
According to the invention, in preferred embodiments, DNA, preferably plasmid DNA, is readily purified, by use of selective precipitation, preferably by addition of compaction agents. Also included is a scaleable method for the liquid-phase separation of DNA from RNA. RNA may also be recovered by fractional precipitation according to the invention.
We have discovered that RNA, commonly the major contaminant in DNA preparations, can be left in solution while valuable purified plasmid DNA is directly precipitated.
Additional aspects of the invention include mini-preps, preferably of plasmid and chromosomal DNA to obtain sequenceable and restriction digestible DNA in high yields in multiple simultaneous procedures.
Still further aspects disclose enhanced stripping of the compaction agent by a stripping method comprising high salt addition and pH shift, and combinations of these techniques.
Also disclosed is a method of assay in which a labeled probe is precipitated when it is hybridized to a target, (e.g. chromosomal DNA, oligonucleotides, Ribosomal RNA, tRNA), and thereafter precipitating the probe/target complex with compaction agents and leaving in solution any unhybridized probe. For example, chromosomal DNA, plasmid, ribosomal RNA, and oligonucleotides can be recovered in excellent purity; by then heating the mixture of nucleic acids and probe (above their melting temperature if the hybridization site is buried within secondary structure) and thereafter precipitating the probe and the target, whereby the target can be detected.
Further disclosed is a method for producing a reduced-viscosity cell lysate, useful as a starting point for further purification of product by removal of nucleic acids through compaction precipitation.
Each of these parameters is discussed below:
A new method for DNA separation has been developed using selective precipitation with small-molecule compaction agents, such as spermine and spermidine, which bind in the grooves of a double-stranded DNA molecule. Compaction precipitation uses compaction agents to neutralize the highly charged phosphate backbone of nucleic acids and to stabilize intermolecular interactions leading to precipitation. This selective precipitation has been demonstrated to separate double-stranded plasmid DNA from RNA, protein and other contaminants in solution. Using compaction precipitation, we have also developed an improved mini-prep procedure capable of producing sequencing-grade plasmid DNA. The precipitation of nucleic acids from lysates can also be applied to the clarification of protein lysates before any subsequent chromatography is done.
In addition a compaction agent-based selective precipitation of RNA from clarified lysates of bacteria, fungi, or metazoan cells and/or mixtures of biomolecules has been developed. The use of selective precipitation with compaction agents and anion-exchange chromatography have been shown to effectively separate the ribosomal RNA""s from each other and 5S rRNA from tRNA. Compaction agent-based separation of RNA produces either a total RNA mixture or a high molecular weight RNA fraction with little contaminating protein or DNA. Anion-exchange chromatography is then used to separate the different RNA molecules from the total bacterial RNA sample. Also, using compaction precipitation and labeled oligonucleotide probes, a hybridization assay has been developed for use in a wide variety of applications, including e.g. environmental monitoring, quality control of nucleic acids, medical diagnostics, and use in mutation studies.
Still another embodiment comprises a method of isolating nucleic acid-binding proteins by coprecipitating them with the nucleic acids to which they bind has been developed. This method can be used in purification and identification of regulatory proteins, histones, and aptamers, for example.
Cell Mass
The starting material is often a mass of cells prepared by fermentation or cell culture, isolated from the environment, or derived from tissues. The cells are then disrupted so the nucleic acids go into solution, forming a lysate. The lysate then optionally undergoes an alkaline lysis or other process to form a clarified lysate. The preferred feed to the compaction precipitation step is a clarified lysate or synthetic mixture. A variety of cell types can be used as feed for this whole process, with bacterial, yeast, other eukaryotic, Gram-negative and Gram-positive being preferred and Gram-negative being most preferred.
Product
The product of the invention can be purified DNA, RNA or nucleic acid-binding proteins, preferably DNA, and most preferably plasmid DNA, e.g. as used in preparation of influenza or other vaccines. Alternative preferred product is RNA, preferably ribosomal RNA, ribozymes, aptamer, artificial RNA, and any other RNA based molecule.
Particularly preferred is RNAse-free plasmid having a quantity of nucleases below current limits of detection and/or low endotoxin contamination. In other embodiments, the product can be a bioassay or protein, e.g. as produced in Examples 13 and 16.
In general, the selective precipitation of the invention can be applied to all bacteria (Gram-negative, Gram-positive and Archaea), all eukaryotes (such as yeast and human cells), recombinant cells, and all synthetic nucleic acids. The invention can separate YAC""s (yeast artificial chromosomes). YACs are very large plasmids in yeast, used in sequencing projects. The invention can also be applied to the production of cosmids (basically very large plasmids in general), and phage and other viral DNA, and the detection of protein-nucleic acid binding and viruses.
Compaction Agents
The compaction agents are preferably small, cationic molecules, which bind in either the major or minor grooves of a double-stranded RNA or DNA molecule, reducing the volume occupied by the nucleic acid. FIG. 1 shows the structures of some common compaction agents). Some compaction agents function in vivo to package genomic DNA into sperm (see reference 7), and can also serve a similar function in the delivery of DNA pharmaceuticals. (See reference 8).
Compaction of DNA involves charge neutralization in combination with stabilization of inter-helix interactions. The compaction agent binds in either the major or minor groove, in proximity to the negatively charged phosphate groups. Precipitation occurs when adjacent DNA helices are affected simultaneously, with the compaction agent not only reducing the helix-helix repulsion but also bridging the helixes. Hoopes described this phenomenon in 1981 (see reference 9) but upon further investigation, we have discovered that RNA is far less readily precipitated by certain compaction agents, preferably linear polyamine type compaction agents, and found that RNA can be selectively precipitated and even fractionated using specialized compaction agents, most preferably, hexammine cobalt as the compaction agent and/or without substantial precipitation of contaminating endotoxins.
In general, there will be added about 0.1 to 20, more preferably about 0.2 to 15 and most preferably about 0.3 to 5 mM of a compaction agent, preferably selected from the group consisting of: basic polypeptides (e.g. polylysine), polyamines (e.g. protamine, spermidine, spermine, putrescine, cadaverine, etc.), trivalent and tetravalent metal ions (e.g. hexammine cobalt, chloropentammine cobalt, chromium (III)), netropsin, distamycin, lexitropans, DAPI (4xe2x80x2,6 diamino 2-phenylindol), berenil, pentamidine, manganese chloride. At present knowledge, the moieties in parenthesis will be more preferred, but any other molecule that can be used to compact DNA via the mechanism described above may be used according to the product to be produced and the cell mass available.
Many other agents may be considered compaction agents and these include: basic polypeptides (i.e. polylysine), polyamines (i.e. protamine, spermidine, spermine, cadaverine, etc.), trivalent and tetravalent metal ions (i.e. hexammine cobalt, chloropentaminie cobalt, chromium (III)), netropsin, distamycin, lexitropans, DAPI (4xe2x80x2, 6 diamino 2-phenylindol), berenil, pentamidine, manganese chloride, or any other molecule that can be used to compact DNA via the mechanism described above (see references 1-7, 9,17-19, 36,37,38). Also any protein having multiple binding domains for nucleic acids can potentially, for large complexes, result in the precipitation of nucleic acids.
Lysing Agents
Lysing agents, preferably detergents, more preferably nonionic detergents, are used to break down cell membranes, thus releasing DNA, RNA and proteins from the cells. The most preferred lysing agent for plasmid DNA is the alkaline lysis detailed in Example 1. The most preferred lysing agent for RNA is Bacterial Protein Extraction Reagent (BPER) which has an unknown composition (it is a proprietary mixture of nonionic detergents marketed by the Pierce Chemical Company), but other nonionic detergents are useful and many detergents are operable, even some anionic and cationic detergents under certain applications. The nonionic detergent lysing agents will generally be added to the cell mass in a concentration of about 0.1 to 5, more preferably 0.5 to 2 wt %. Other known lysing agents can also be used with the technology such as freeze/thawing, French cell press, enzymes, microfluidization, sonication, etc.
Nucleases
One of the main advantages of the compaction precipitation technology is that it circumvents the need to use nucleases, proteases or carbohydrases. Selective precipitation directly harvests nucleic acids and the target nucleic acid of a precipitation can be changed by changing conditions (i.e. type of compaction agent, quantity of compaction agent, concentration of salts, etc.) Because of this selectivity other large biomolecular contaminants such as proteins, unwanted nucleic acids, carbohydrates, etc. do not have to be degraded by enzymes. Thus the use of RNAse, DNAse, proteases, and other enzymes is unnecessary.
pH
All Examples are carried out at a pH between 6-8, to keep nucleic acid degradation to a minimum, though other pHs may be preferred in certain cases. The compaction agents can be affected by extreme pH. In fact, we have found that pH change (e.g., shifting the pH past the pKA of the amine groups in polyamines, so that they lose their positive charge and do not bind nucleic acids strongly is one of the ways to separate nucleic acids from the compaction agents themselves.)
Ionic Strength
High ionic strength can negate the effects of compaction agents. The preferred maximum ionic strength for compaction precipitation is 250 mM NaCl when plasmid is precipitated in 10 mM spermine. More preferred ionic strength before compaction agent addition is about 0-50 mM, more preferably 1 to 20 mM but those skilled in the art will adjust the ionic strength to best suit the particular lysate and compaction agents being employed. Changing ionic strength is an easy way to separate the compaction agents from the nucleic acids, because in the presence of a high ionic strength solution the compaction agents are displaced from the nucleic acid backbone.
Hybridizing
To hybridize means to bind to its complementary sequence in the target. If the probe used in a bioassay includes a sequence 5xe2x80x2-AAGC-3xe2x80x2; its hybridizing complementary sequence will be 5xe2x80x2-GCTT-3xe2x80x2. This is important because this test can be run as a valuable quality control measure on oligonucleotides and other synthetic nucleic acids, or used for detection of particular nucleic acid sequences and/or viruses in cells or tissues.
Batch or Continuous Conditions
The invention can be performed in commercially available equipment under batch or, less preferably, continuous flow stream, conditions; at elevated, reduced or atmospheric pressure and temperature, but atmospheric pressure and near ambient temperatures will be preferred for most applications.
Most large-scale bioseparations are done in batch because of the need to grow cells and the difficulty of maintaining a steady flow of cells from a chemostat, also the preparation will preferably be conducted under 50 degrees C. and more preferably under 25 C.
The present invention is useful in the separation of DNA from RNA and vice versa. With numerous gene therapy products entering clinical trials, new and innovative strategies are needed to produce pure plasmid DNA.
In addition with the advances in gene chips in which DNA is attached to a small piece of glass (so that one chip can have over 1 million nucleic acid probes and can be used to test for disease) and genetic diagnostics, environmental monitoring, ribozyme research, and aptamers, improved separation processes for nucleic acid molecules are in demand.
The separation of RNA from bacterial cells is conventionally achieved by phenol/chloroform extraction and polyacrylamide gel electrophoresis. However, this conventional use of organic solvents and polyacrylamide (a neurotoxin) creates hazardous waste, and this approach is not easily scaleable for medium to large-scale production of RNA.
Selective precipitation by use of compaction agents according to the present invention provides lower cost, more effective and faster separation than the conventional methods of plasmid production. (See references 10 and 14) An added unexpected advantage of the selective precipitation of the invention is that it also contributes to improved performance of subsequent chromatographic columns used for further separation and purification.
Of considerable value in production of pharmaceuticals, the invention permits the precipitation of plasmid DNA containing less than 0.3 Units endotoxin per microgram plasmid DNA EU/xcexcg or IE/xcexcg).